Novel drugs, targeting virus entry and maturation, have recently been added to the growing armory of antiviral agents aimed at stemming the continuing spread of HIV infection. In addition, host factors participating in key steps in HIV replication are under consideration as targets for therapeutic intervention. Despite these promising approaches, the viral enzymes, protease (PR), reverse transcriptase (RT) and integrase (IN), remain primary targets of highly active antiretroviral therapy, with almost 20 drugs in use against PR and RT, and IN inhibitors undergoing clinical trials. With respect to HIV-1 RT, our section combines biochemistry and biophysics with structural, molecular, and chemical biology to provide molecular details of its role in converting RNA of the invading virus into integration-competent double-stranded DNA. Using nucleoside and amino acid analogs to provide novel acumen into the structures of complexes representing replication intermediates complements traditional nucleic acid and protein mutagenesis methods. Chemical nucleic acid footprinting is now complemented with direct mass spectrometric analysis, while single-molecule fluorescence studies have been introduced to probe the orientation of RT on a variety of nucleic acid duplexes. In this project, we have investigated structural features of the polypurine tract (PPT) primer of (+) strand DNA synthesis mediating its recognition by the RNase H domain of HIV-1 RT. Such studies indicated that template nucleobases can be removed without loss of cleavage specificity at the scissile -1/+1 phosphodiester bond, while equivalent lesions in the RNA primer are inhibitory. Subsequent studies identified individual bases of the PPT primer that are critical to its function. The use of purine analogs has also allowed us to determine the role of the exocyclic groups of the purine ring at these critical positions. Finally, using a combination of analog-substituted substrates and mutants of HIV-1 RT, we have used nucleoside analogs to understand how the single-stranded template interacts with residues of the fingers subdomain prior to access the DNA polymerase active site of HIV-1 RT. In collaboration with the NCI/SAIC Protein Expression Laboratory, we coupled a cell-free translation system with a novel suppressor tRNA technology to site-specifically insert unnatural amino acid analogs into the p66 subunit of p66/p51 HIV-1 RT (Sitaraman et al., 2003). Using this approach (Klarmann et al., 2004), we substituted m-fluoro-Tyr and nor-Tyr for Tyr183 of the DNA polymerase -Tyr-Met-Asp-Asp- active site motif, the latter of which resulted in the loss of RNA-dependent DNA polymerase activity while DNA-dependent DNA polymerase activity was unaffected. In a subsequent study, we evaluated five HIV-1 RT variants containing tyrosine analogs at position 115 of their p66 subunit. Each mutant retained significant DNA polymerase activity, and two were selected for detailed kinetic analysis. Aminomethyl-Phe115 RT incorporated dCTP more efficiently compared with wild-type RT and was resistant to the chain-terminating nucleoside analog (-)-beta-2',3'dideoxy-3'-thiacytidine triphosphate (3TCTP). 2-Naphthyl-Tyr115 RT activity was significantly impaired at low dCTP concentrations and was kinetically slower with all dCTP analogs tested. Structural models of HIV-1 RT ternary complexes containing these amino acid substitutions have revealed probable mechanisms of the observed catalytic rate changes. Finally, genetically manipulated E. coli strains have been used to site-specifically introduce photoactivable amino acids for crosslinking to a variety of biomolecules. As a result of this work, an Employee Invention Report on "Non-covalent attachment of fluorophores to oligonucleotides for high throughput sequencing" (Inventors Jason W. Rauch and Stuart J. Le Grice) was submitted in 2009. [Corresponds to Le Grice Project 1 in the April 2007 site visit report of the HIV Drug Resistance Program]